The time needed to find a visual target amongst distractors (search task) can increase as a function of the distractors’ number (set-size) in the search-array (inefficient search). While the allocation of attention in search tasks has been extensively investigated and debated in the visual domain, little is known about these mechanisms in touch. Initial behavioral evidence shows inefficient search behavior when participants have to distinguish between target and distractors defined by their vibro-tactile frequencies. In the present study, to investigate the allocation of attention to items of the search-array we measured the N140cc during a tactile task in which the set-size was manipulated. The N140cc is a lateralized component of event-related brain potentials recently described as a psychophysiological marker of attentional allocation in tactile search tasks. Participants localized the target, a singleton frequency, while ignoring one, three or five homogeneous distractors. Results showed that error rates linearly increased as a function of set-size, while response times were not affected. Reliable N140cc components were observed for all set-sizes. Crucially, the N140cc amplitude decreased as the number of distractors increased. We argue that the presence of additional distractors hindered the preattentive analysis of the search array resulting in increased uncertainty about the target location (inefficient preattentive stage). This, in turn, increased the variability of the deployment of attention to the target, resulting in reduced N140cc amplitudes. Consistent with existing behavioral evidence, these findings highlight systematic differences between the visual and the tactile attentional systems. These mechanisms are typically investigated in search tasks in which participants have to identify the presence (or a feature) of a task relevant item (target) presented simultaneously with a number of irrelevant ones (distractors). Search efficiency is typically assessed by manipulating the number (set-size) and features of distractors presented with the target. Efficient search behavior is unaffected by set-size, as indicated by a flat slope function between performance indicators (response times and/or accuracy) and set-size and is usually observed when target and distractors can be distinguished on the basis of a distinctive feature e.g., . The preWhile most studies on search behavior involved visual search tasks, researchers have started to address analogous questions in the tactile domain. When participants were allowed to haptically explore the tactile stimuli presented to their fingers, results revealed that search efficiency depended on the specific dimension defining target and distractors c.f. . EfficieOther researchers have exploited the ability of the somatosensory system to discriminate the frequencies of different vibrotactile stimuli e.g., . Groen eEvent-related potential (ERP) studies have recently identified the electrophysiological correlate of target selection in tactile search tasks e.g., , 2022. EIn a series of recent tactile search ERP studies, participants were asked to localize a target presented simultaneously with one salient but irrelevant singleton distractor and with several other homogeneous distractors , 2022. ONotably, existing evidence has also shown potentially relevant differences between the mechanisms responsible for visual and tactile target selection. In the visual domain, the interference created by a singleton distractor increased as the distance between target and distractor decreases within the same hemifield, as revealed by worst performance and reduced N2pc components for contiguous singletons, due to the progressively overlapping neural resources assumed to represent these items e.g., . In toucThe studies reviewed above can be considered as a first attempt to characterize the functional meaning of the N140cc component and the mechanisms responsible for the selection of relevant information in touch. However, basic questions related to search efficiency and its impact on the N140cc component remain completely unexplored. In the visual modality, different search tasks have yielded different pattern of N2pc modulations by set-size, with some studies showing a decreasing N2pc with increasing distractors e.g., , while oMage = 21 years, SDage = 3.6 years; 27 right handed and 1 ambidextrous, A total of thirty-seven volunteers were recruited via word-of-mouth at the University of Modena and Reggio Emilia. All of them had a normal or corrected-to-normal vision and no history of neurological disorders. Three participants were excluded from the analyses due to low accuracy levels in the behavioral task . Following the ERP data processing procedure, six participants’ data were excluded due to a low number of trials . A total of 28 participants remained in the sample  Ethics Committee and followed the Helsinki Declaration principles. All participants signed an informed consent before starting the experiment.Participants placed their hands on a table with their palms down. Tactile stimuli were presented using 12 V solenoids  driving a metal rod with a blunt conical tip. The tip of the tactile stimulators touched the skin whenever a current passed through the solenoid. Six tactile stimulators were used in total, each one was attached with adhesive medical tape to the inner side of the top phalanx of the left and right index, middle and ring fingers. The distance between the index fingers of the two hands was 10 cm. Once in the correct position, the hands were covered with a black cardboard, on top of which there was a white pin aligned with the body midline which served as fixation-point. To mask the sounds made by tactile stimulators, one speaker was positioned on the table close to the hands and presented white noise (65 dB SPL) throughout the experimental blocks. Two vertically arranged foot-pedals  were positioned under the toes and heel of one of the participants’ feet. Participants were asked to keep one foot on these response pedals during the task.On each trial, a search-array was presented to mirror-symmetric (homologous) fingers of the left and right hand. There were three different types of search-arrays in which two, four or six vibro-tactile stimuli were presented simultaneously  (405 ms duration). The search-array presentation was followed by a 1,800 ms interval which was used to collect foot responses. Thus, the interstimulus interval between search-arrays was set at 2.505 ms.Ten blocks of 72 trials each were completed by participants. The tactile target was presented randomly and with equal probability to the index, middle or ring finger of the left or right hand . Within each block of trials, 2-, 4-, and 6-items search-arrays were equally likely .Participants’ task was to identify the location of the target (left or right hand) by pressing the top or bottom pedal with their toes or heels. They were instructed to keep their eyes on the central fixation at all times and to press the pedals as fast and as accurately as possible. To eliminate lateralized motor activity in the grand averaged ERP waveforms, participants completed five successive blocks responding with their right foot, and the remaining five blocks with their left foot. The order of the responding foot was counterbalanced across participants.Prior to the beginning of the experiment participants completed two 36-trials practice blocks (which was repeated whenever average accuracy fell below 60% during this training), following a familiarization procedure with the stimuli frequencies. At the end of each block participants received verbal feedback about their performance (average response time and accuracy).Electroencephalography (EEG) was recorded with a BrainAmp amplifier system (500 Hz sampling rate) from 64 active electrodes positioned according to 10–20 system. EEG data was analyzed using Brain Vision Analyser (version 2.0.4.368). EEG was digitally re-referenced to the average of the left and right earlobe and was digitally filtered offline . The EEG was epoched into 450 ms intervals starting 100 ms before and ending 350 ms after the search-array onset. Trials with eye blinks, horizontal eye movements and other artifacts (voltage exceeding ± 80 μV at any electrode sites) were excluded from further analysis, as were trials with response errors. Participants with less than 60% of the trials in at least one of the set-size conditions were excluded from the analyses. This led to the exclusion of 6 participants. The average number of trials included in each condition for the remaining 28 participants was 185 for the 2-items array , 167 for the 4-Items (70%) and 150 for the 6-Items (63%).t-tests) between these difference waveforms observed for the different set-sizes (Bonferroni corrections were applied to these multiple comparisons). Finally, to test whether the N140cc amplitude was linearly related to the set-size of the search-array, polynomial orthogonal contrasts were carried out.Event-related potentials elicited by the presentation of the search array on correct trials were averaged relative to a 100 ms pre-stimulus baseline separately for all combinations of set-sizes (2-Items vs. 4-Items vs. 6-Items search-array) and target side (left vs. right hand). The N140cc was quantified for each participant and for each set-size on the basis of ERP mean amplitudes obtained at lateral central electrodes C3/4 and C5/6 . Mean reaction times (RTs) as well as accuracy rates were submitted to separate repeated measure ANOVAs with set-size (2 Items vs. 4 Items vs. 6 Items search-array) as within-subjects factor. Following set-sizes main effects, we run contrasts between the means observed for the different set-sizes (Bonferroni corrections were applied to these multiple comparisons). Similarly to the ERP analysis, polynomial orthogonal contrasts were also carried out to investigate whether mean RTs and accuracy rates were linearly dependent on set-size.p-values were reported.For all analyses, Greenhouse-Geisser adjustments to the degrees of freedom were applied where appropriate, and unadjusted F = 95.9, p < 0.001, ηp2 = 0.78, with participants’ performance being most accurate in the 2 Items array , intermediate in the 4 Items array  and worst in the 6 Item arrays  . Accuracy rates linearly decreased with increasing set-sizes, F = 144, p < 0.001, ηp2 = 0.84.For the accuracy data  = 1.7, p = 0.189, ηp2 = 0.06, see Results of the ANOVA carried out on RTs revealed no significant effect of set-size, F = 1.06, p = 0.35, ηp2 = 0.038, suggesting no differences between ERPs elicited by the different set-sizes. The main effect of the factor laterality, F = 18.2, p < 0.001, ηp2 = 0.43, revealed the presence of a reliable lateralized component between 70 and 100 ms regardless of set-size. This was further modulated by set-size , F = 5.19, p < 0.013, ηp2 = 0.16. Pair-wise comparisons between ipsilateral and contralateral ERP amplitudes carried out separately for each set-size revealed the presence of reliable lateralizations for the 2-items array  and 4-items array  but not for the 6-items array . The amplitude of this early lateralized component linearly decreased as a function of set-size . Bonferroni-adjusted contrasts carried out between the amplitudes observed for the different set-sizes showed a larger lateralized component for 2- Items arrays compared to 4- and 6-Items arrays (both p < 0.033). However, no statistical difference emerged between 4- and 6-Items arrays (p = 0.9).An exploratory analysis of the mean amplitude values measured between 70 and 100 ms post array onset showed that the main effect of set-size was not statistically significant, F = 5.6, p < 0.006, ηp2 = 0.17, that was driven by the fact that ERP amplitudes increased with set-size regardless of laterality. The N140cc component was reliably present between 110 and 250 ms  = 40.02, p < 0.001, ηp2 = 0.6). Crucially, the interaction of interest between laterality and set-size was significant, F = 13.1, p < 0.001, ηp2 = 0.33. Pairwise comparisons between ipsilateral and contralateral ERP amplitudes carried out separately for each set-size revealed the presence of reliable lateralized N140cc components for each set-size  > 2.9, p < 0.007, d > 0.055). The amplitude of the N140cc component linearly decreased as a function of set-size, F = 17.6, p < 0.001, ηp2 = 0.39. Bonferroni-adjusted contrasts carried out between the N140cc amplitude observed for the different set-sizes showed a larger N140cc for 2- Items arrays compared to 4 and 6 Items arrays (both p < 0.001). However, no statistical difference emerged between 4 and 6 Items arrays (p = 0.6).The planned analysis of mean amplitude values  revealed a significant main effect of set-size, To evaluate the time course of the N140cc we computed the mean amplitude values of the waveforms ipsilateral and contralateral to the target separately for each set-size in consecutive 20 ms intervals from 90 to 310 ms see . ReliablWithin the more general purpose of understanding similarities and differences between the neural mechanisms responsible for the allocation of attention in the visual and tactile sensory domains, the aim of this study was to investigate whether the amplitude of the N140cc component elicited during a tactile search task is modulated by set-size. Participants showed inefficient search behavior, as indicated by the fact that accuracy rates linearly decreased as a function of distractors’ number, although RTs were not affected by set-size. The N140cc component was reliably present contralateral to the target side regardless of the number of distractors. Crucially, the amplitude and time course of the N140cc component was modulated by set-size. The N140cc amplitude was maximal and emerged in earlier time-windows in the 2-items array and decreased linearly as the number of distractors increased.According to 1. Thus, the present ERP findings show inefficiencies at both the preattentive and attentive stages of processing2.In the present study, the number of distractors reduced the accuracy of target localization showing that the target did not pop out from the array despite being more salient than the homogenous distractors c.f. . This isIntriguingly, in addition to the N140cc components, an early lateralization was elicited between 70 and 100 ms over central electrodes and modulated by set-size, with decreasing amplitudes for larger set-sizes. We speculate that this early component may reflect the efficiency of the preattentive analysis, similarly to the PPC component observed in visual search tasks when a singleton item is presented amongst homogeneous distractors e.g., . If thisIt is worth noting that the increased perceptual noise experienced by participants with larger set-sizes may be due - at least in part - to effects of tactile masking which are known to cause distortions in the sensory representation of the tactile stimulus e.g., . In the duration of the N2pc  Ethics Committee. The patients/participants provided their written informed consent to participate in this study.EG ideated the study. SR, CI, and FF contributed to the design and implementation of the research. FF programmed the experiment and collected the data. EG and FF analyzed the data and wrote a first draft of the study. CI and SR reviewed and edited the manuscript. All authors provided critical feedback, helped shape the research and the manuscript, and read and agreed to the published version of the manuscript.


This study aimed to evaluate the effectiveness of various scoring systems in predicting in-hospital mortality for COVID-19 patients admitted to the internal medicine ward. We conducted a prospective collection of clinical data from patients admitted to the Internal Medicine Unit at Santa Maria Nuova Hospital in Florence, Italy, with confirmed pneumonia caused by SARS-CoV-2. We calculated three scoring systems: the CALL score, the PREDI-CO score, and the COVID-19 in-hospital Mortality Risk Score (COVID-19 MRS). The primary endpoint was in-hospital mortality. : A total of 681 patients were enrolled in the study, with a mean age of 68.8 ± 16.1 years, and 54.8% of them were male. Non-survivors had significantly higher scores in all prognostic systems compared to survivors . The receiver operating characteristic (ROC) analysis yielded the following area under the curve (AUC) values: MRS 0.85, CALL 0.78, PREDI-CO 0.77. The addition of Delirium and IL6 to the scoring systems improved their discriminative ability, resulting in AUC values of 0.92 for MRS, 0.87 for CALL, and 0.84 for PREDI-CO. The mortality rate increased significantly across increasing quartiles (p<0.001). In conclusion the COVID-19 in-hospital Mortality Risk Score (MRS) demonstrated reasonable prognostic stratification for patients admitted to the internal medicine ward with SARS-CoV-2-induced pneumonia. The inclusion of Delirium and IL6 as additional prognostic indicators in the scoring systems enhanced their predictive performance, specifically in determining in-hospital mortality among COVID-19 patients. In December 2019, a new coronavirus family member named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was discovered in Wuhan, Hubei, China. Over the following 2 years, it rapidly spread worldwide, leading to a pandemic.COVID-19 exhibits a wide range of signs and symptoms, varying from asymptomatic cases to severe pneumonia. Approximately 20% of patients experience acute respiratory distress syndrome (ARDS) and multi-organ failure (MOF)  vs. 9 [7–11], p < 0.001, PREDI-CO score 4 [3–6] vs. 2 [1–4], p < 0.001, MRS score 13 [12–15] vs. 10 [8–12], p < 0.001. To assess the prognostic stratification ability, ROC curve analysis was conducted. The results showed that the MRS score had the highest predictive ability for in-hospital mortality , followed by the CALL score  and the PREDI-CO score  We performed recalculations of the CALL, PREDI-CO, and MRS scores by incorporating the delirium variable. The delirium variable was assigned a score of 3 for the CALL score, 1 point if absent, 3 for the MRS score (1 if absent), and 1 for the PREDI-CO score (0 if absent) based on the OR values from our dataset.The scores, when calculated with the addition of delirium, demonstrated improved performance compared to the original scores see Fig. d–f: MRS IL-6 levels were divided into tertiles, and a different score was assigned to each tertile. For the MRS score, 1 point was assigned for IL-6 < 17.1, 2 for 17.1 ≤ IL-6 ≤ 48.8, and 3 for IL-6 > 48.8. Each tertile with a higher IL-6 value was associated with a statistically significant increase in mortality compared to the previous tertile (p < 0.001). Regarding the CALL score, a score of 1 was assigned to values lower than 17.1, while a score of 3 was assigned for values ≥ 17.1. In the first group, the percentage of deaths was 1.9% compared to 15% in the second group (p < 0.001). Lastly, for the PREDI-CO score, values lower than 17.1 were assigned a score of 0, while values ≥ 17.1 were assigned a score of 1. In the first group, the percentage of deaths was 1.9% compared to 15% in the second group (p < 0.001).Upon recalculation of the scores with the inclusion of IL-6, the following results were obtained Fig. g–i: MRS By incorporating IL-6 and delirium into the scores, the highest level of performance was achieved. The scoring system for IL-6 and delirium remains as described earlier. The results are as follows Fig. j–l: MRS Finally, we assessed the mortality rate across different quartiles of the scores  and laboratory values commonly used in clinical practice . The scoOn the other hand, this study represents the initial attempt at external validation of the PREDI-CO score and COVID-19 in-hospital Mortality Risk Score (COVID-19 MRS). The MRS score was validated in a small Italian geriatric population of 221 consecutive patients with COVID-19 aged ≥ 75 years admitted to two hospitals . Our resCognitive impairment was frequently observed in our patient sample, with an overall prevalence of 18%. It ranked as the fourth most common comorbidity after hypertension, diabetes mellitus, and cardiovascular disease. This observation aligns with a study conducted on 1970 patients hospitalized for COVID-19 pneumonia in a Spanish tertiary hospital . RegardiCognitive impairment primarily affects the elderly population, who often possess intrinsic physiological frailty and numerous comorbidities that independently contribute to the risk of death. Dementia is frequently associated with the presence of the apolipoprotein E4 gene, which is widely expressed in lung tissue and may predispose individuals to an exaggerated inflammatory response, potentially facilitating the onset of acute respiratory distress syndrome (ARDS) . SeveralIn our patient sample, elevated levels of interleukin 6 (IL-6) measured upon hospital admission were associated with an increased risk of death. These findings align with existing literature. Grifoni et al.  and a meDelirium emerged as the most frequent complication in our population, affecting 61 patients (approximately 10% of the population). Two different meta-analyses by Shao et al.  and PranBased on these results, the addition of delirium as an additional evaluation factor improves the performance of each prognostic score. While IL-6 augmentation enhances the predictive power of clinical scores, delirium alone enhances the performance of COVID-19 clinical scores and may offer a faster and more cost-effective approach compared to measuring IL-6 levels. In conclusion, incorporating delirium into COVID-19 clinical scores enhances their performance, and although IL-6 remains a valuable predictor, delirium alone may offer a more practical and cost-effective approach. Continuous research and evaluation of different predictors are crucial for developing accurate and efficient clinical scoring systems for predicting outcomes in COVID-19 patients.The single-center design of the present study is a noteworthy limitation. Consequently, the generalizability of these results may be compromised due to variations in local admission and management policies. The implementation of a standardized data collection template restricted the potential for subjective data interpretation. Moreover, the observational nature of our analysis prevents us from drawing definitive conclusions regarding the clinical factors influencing mortality and their associations with therapeutic strategies, which were also subject to adaptations over time.Prognostic scores serve as valuable tools for stratifying the prognosis of patients with COVID-19 who are admitted to internal medicine wards. These scores have been examined and have demonstrated a strong predictive capability for hospitalized patients. Additionally, it has been observed that delirium acts as a predictor of in-hospital mortality, specifically in elderly patients with COVID-19 pneumonia. The incorporation of delirium into the existing three prognostic scores has been found to enhance their prognostic value.Scores, as zero-cost tools, play a crucial role in our day-to-day lives by aiding in the effective stratification of our patients. It is imperative that we make use of these valuable tools to enhance our understanding of patient outcomes and guide appropriate interventions.


CNI-based IS maintenance throughout the dialysis period after KTF may improve retransplantation outcomes.The impact of immunosuppressive therapy (IS) strategies after kidney transplant failure (KTF) on potential future new grafts is poorly established. We assessed the potential benefit of calcineurin inhibitor (CNI)-based IS maintenance throughout the dialysis period on the outcome of the second kidney transplant (KT). We identified 407 patients who underwent a second KT between January 2008 and December 2018 at four French KT centers. Inverse probability of treatment weighting was used to control for potential confounding. We included 205 patients with similar baseline characteristics at KTF: a total of 53 received at least CNIs on the retransplant day (G-CNI), and 152 did not receive any IS (G-STOP). On the retransplant date, G-STOP patients experienced a longer pretransplant dialysis time, were more often hyperimmunized, and underwent more expanded-criteria donor KTs than G-CNI patients. During the second KT follow-up period, rejection episodes were similar in both groups. The 10-year survival rates without death and dialysis were 98.7% and 59.5% in G-CNI and G-STOP patients, respectively. In the multivariable analysis, CNI-based IS maintenance was associated with better survival (hazard ratio: 0.08; 95% confidence interval: 0.01–0.58,   Since the 2000s, the number of patients waiting for a second transplant after kidney transplant failure (KTF) has increased year after year. Currently, they represent 13%–23% of patients on the waiting list  and appThe optimal management of IS after KTF in potential candidates for a second kidney transplant (KT) remains uncertain . Until rThe objective of the present retrospective, multicenter, observational study was thus to evaluate the impact of CNI-based IS maintenance during the dialysis period until the new transplantation on the outcome of the second graft.This retrospective, multicenter study was performed at four French adult KT centers . Patients were selected using the Cristal prospective database. The inclusion criteria were patients over 18 years old who had undergone a second KT between 1 January 2008 and 31 December 2020 at the Clermont-Ferrand, Rouen, or Poitiers transplant centers or between 1 January 2016 and 31 December 2020 at the Bordeaux transplant center (because of a change of the computerized patient record systems). The exclusion criteria consisted of second preemptive transplantations, continuation of IS treatment without CNIs, and multiorgan transplantations.The following demographic, clinical, and biological data were collected: i) at the time of KTF—age, sex, body mass index, initial kidney disease, first transplant outcome and cause of allograft failure, PRA level, and the eventual presence of donor-specific anti-HLA antibodies (DSAs); ii) at the inscription on the waiting list for a second KT—PRA level and comorbidities ; iii) during the dialysis period—potential allograft nephrectomy, severe infection defined as an opportunistic infection  or requiOversight and study approval were provided by the Committee for Protection of Human Subjects (CPP SUD-EST VI) on 3 September 2019  and by the National Consultative Committee on the Use of Health Research Information (14.510). No written consent was required for this study, but a non-opposition letter was sent to all patients in accordance with national legislation (MR-004 reference methodology) .Two groups of patients were defined according to the modality of management of IS in the period between the two KTs: i) the CNI group (G-CNI), defined by the continuation of IS including CNIs either as monotherapy or in combination with other IS  during the entire period between the two KTs, and ii) the stop group (G-STOP), defined by the cessation of all IS during the intertransplant period.Statistical analyses were performed with Stata software . All tests were two-sided with an alpha level set at 5%. Categorical variables were expressed as number of patients and associated percentages, and continuous variables as mean ± standard deviation or median , according to their statistical distribution.Demographic and first transplant characteristics were compared between G-STOP and G-CNI using usual statistical tests: chi-squared test or Fisher’s exact test for categorical variables and Student’s t-test or Mann-Whitney test for continuous variables.To assess the relationship between the group (G-STOP and G-CNI) and the primary and secondary endpoints, a propensity score (PS) analysis was implemented using the inverse probability of treatment weighting (IPTW) method , 38. Thep < 0.10) as well as their clinical relevance14,32. The results are expressed as hazard ratios (HRs) and 95% confidence intervals (CIs).The primary outcome was a composite of dialysis and death after the second KT, presented as survival free of dialysis and death. This outcome was expressed as censored data and was estimated with the Kaplan-Meier method, and the groups were compared by the log-rank statistic. Multivariable analyses were performed with a Cox model (with the center as a random effect) considering covariables in terms of their significant results in univariate analysis  over the total duration of follow-up and are expressed per 100 patient-years (p-y).Among the 3246 KTs performed at the four centers during the study period, 407 patients (12.5%) received a second KT . Five pap = 0.02), a shorter transplantation survival , and a higher cPRA at inscription on the waiting list for retransplant . The rate of hyperimmunized patients (cPRA ≥85%) in the G-STOP and G-CNI was 26.7% and 13.2%, respectively (p = 0.04). Among G-CNI patients, 36 (61.0%) were treated with tacrolimus and 23 (39.0%) with cyclosporine. IS maintenance until the second KT consisted of CNI monotherapy in 19 patients (32.2%) and CNI combined with an antimetabolite or corticosteroid therapy in 30 patients (50.8%). Ten patients (17.0%) received triple IS.The characteristics of the patients before IPTW are depicted in The characteristics at the time of KTF of the 205 patients included in the PS analysis are summarized in p < 0.001). The waiting times from relisting to the second KT were 16 months  in the G-CNI and 27 months  in the G-STOP (p = 0.06)  .p = 0.001). The rate of hyperimmunized patients was also lower in the G-CNI: 23.9% versus 55.2% in the G-STOP (p < 0.001). The numbers of patients transplanted with preformed DSAs and induction treatment were comparable between the groups . Hyperimmunized patients, compared with patients with cPRA levels <85%, were more likely to receive a kidney transplant from an expanded criteria donor . On the day of the second KT, 44.1% of G-CNI patients had a residual diuresis ≥500 mL compared to 13.8% in G-STOP (p = 0.06). The delayed graft function rate was 9.9% in the G-CNI and 25.8% in the G-STOP (p = 0.001). The numbers of patients transplanted with preformed DSA and an induction treatment were comparable between the groups  (p = 0.01) (p = 0.001). Data on survival before IPTW are presented in After the second KT, 10 years survival free of dialysis and death was significantly better in the G-CNI than in the G-STOP  , with 10 = 0.01) . The difp = 0.004). The main cause of graft loss was rejection (45.1%). The number of humoral rejections and the occurrence of DSA were comparable in the two groups, but there was less cellular rejection in the G-CNI than in the G-STOP . All deaths were observed in the G-STOP  in the G-CNI and G-STOP were similar . The ratp = 0.04) (p = 0.02)  . The ser = 0.04) , but the = 0.02) .p < 0.001) but a lower rate of major cardiovascular events . The exposure-adjusted rate of neoplasia was similar in both groups  [p < 0.001) [23. The significantly higher rate of hyperimmunized patients in the G-STOP group may explain in part why these patients received more expanded criteria deceased donor allografts [In the literature, the negative impact of the dialysis waiting time after KTF on the subsequent KT outcome and increased mortality is well documented , 14, 21.< 0.001) . In anot< 0.001) . The latlografts . Indeed,lografts –41 and dlografts , whetherde novo DSAs were similar in the two groups, which may explain the similar humoral rejection rates in the two groups [p = 0.02) in patients with in situ previous transplants who also usually had CNI maintenance compared to patients with first allograft nephrectomy who also usually discontinued therapy. The authors observed less T-cell alloreactivity measured by ELISPOT assay against the pretransplant donor in the group with CNI maintenance for a prolonged period compared to patients with discontinued treatment due to transplantectomy [While the rate of humoral rejection was similar in both groups, we observed a lower rate of cellular rejection in the G-CNI than in the G-STOP after the second KT. The rates of second transplants with preformed DSAs and o groups . Healthyo groups . We can o groups , 43 acquo groups . Indeed,o groups  reportedntectomy .n = 52) and in patients (n = 50) in whom IS was continued with antimetabolites and/or CNIs [The benefit-risk balance of IS maintenance until a new KT is widely debated in the literature. Some retrospective cohort studies have observed higher rates of major cardiovascular, infectious, or tumor events in patients with IS maintenance , 23. How/or CNIs . A Canad/or CNIs . HoweverThe current work includes several limitations. First, due to the retrospective nature of the study, major differences between the two groups were observed, such as the rates of diabetes at the end of the first KT, persistent infections at the time of KTF, and PRAs level at relisting in the G-CNI. We thus proposed a PS analysis using the IPTW method to reduce the effect of these confounding factors that may have influenced survival. However, we cannot exclude the existence of factors not accounted for . Indeed,Our study shows that after KTF, maintaining CNI-based IS in a cohort of patients without heavy comorbidities may reduce the risk of immunization, shorten the waiting time, and provide better access to standard criteria donor grafts. These strategies may improve the survival of the subsequent graft and these patients.


Cannabis sativa L. may provide a good source of anti-inflammatory molecules. Here, we tested the anti-inflammatory properties of cannabis extracts and pure cannabinoids in lipopolysaccharide (LPS)-induced inflammation in human THP-1 macrophages. We found that pre-treatment with cannabidiol (CBD), delta-9-tetrahydrocannabinol (THC), or extracts containing high levels of CBD or THC reduced the level of induction of various cytokines. The CBD was more efficient than THC, and the extracts were more efficient than pure cannabinoids. Finally, IL-6, IL-10, and MCP-1 cytokines were most sensitive to pre-treatments with CBD and THC, while IL-1β, IL-8, and TNF-α were less responsive. Thus, our work demonstrates the potential of the use of cannabinoids or/and cannabis extracts for the reduction of inflammation and establishes IL-6 and MCP-1 as the sensitive markers for the analysis of the effect of cannabinoids on inflammation in macrophages.Inflammation is the response of the innate immune system to any type of injury. Although acute inflammation is critical for survival, dysregulation of the innate immune response leads to chronic inflammation. Many synthetic anti-inflammatory drugs have side effects, and thus, natural anti-inflammatory compounds are still needed.  C. sativa plant contains many metabolites, but the most well-known are cannabinoids, terpenes, and flavonoids. Cannabinoids act via the endocannabinoid system (ECS), which consists of receptors such as cannabinoid 1 (CB1) and cannabinoid 2 (CB2), transient receptor potential channels of the vanilloid subtypes 1 and 2 , G protein-coupled receptors 18, 55, and 119 , endocannabinoids such as 2-arachidonoylglycerol and anandamide , and their biosynthesis and degradation enzymes. Homeostasis maintenance is the main function of ECS [C. sativa plants are cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). The effect of cannabis extracts is frequently different from that of single cannabinoids, with extracts often being more efficient due to the complex interaction between minor cannabinoids, flavonoids, and terpenes—this is known as an entourage effect [The positive impact of cannabis on human health has been known for many years . The C. e effect . The maie effect ,5. ThereInflammation is the body’s vital defense mechanism. Epithelial cells and macrophages are often the first line of defense against bacteria, viruses, and toxins . BacteriThe anti-inflammatory effects of cannabis extracts, as well as their active ingredients, are well documented . For insIn this work, we attempted to study the effects of cannabis extracts on LPS-induced cytokine release in human THP-1 leukemia monocytes. We aimed to establish a model of the induction of inflammation and hypothesized that cannabinoids would prevent a strong induction of inflammation. We further hypothesized that extracts differ from pure cannabinoids in their anti-inflammatory properties. Here, we found that inflammation was induced efficiently at 3 h after application of LPS and was partially prevented by exposure to CBD and various cannabis extracts. We also noted that IL-6, IL-10, and MCP-1 were more sensitive to pre-treatments with cannabinoids as compared with IL-1β, IL-6, and TNF-α.We first tested the CBD and THC concentrations of three extracts we planned to use in this study. We found that extracts #14 and #178 were high in THC, while #131 was a high CBD extract . We alsoWe hypothesized that terpene composition and concentration may affect anti-inflammatory properties and thus tested the terpene profile . CultivaTo establish the inflammation model, we tested the time of induction of pro-inflammatory cytokines. We exposed human THP-1 macrophages to LPS for 3 and 6 h; we used 0.5 μg/mL of LPS as this concentration was shown to be effective in inducing inflammation in cell models . We thenAfter determining that 3 h is the best time for the induction of inflammation by the LPS, we attempted to find out which sample was better for the analysis: the cell culture supernatant (media) or the cell lysate. We exposed cells to LPS for 3 h, harvested media and cell lysates separately, and performed a multiplex ELISA experiment. We found that LPS media samples (3 h of treatment) had much higher levels of IL-1β, TNF-α, and IL-10 cytokines in comparison with LPS cell lysate samples . It was To test the effect of cannabis extracts and pure cannabinoids, CBD and THC, on the reduction of inflammation triggered by LPS, we first tested the viability of THP-1 macrophages in response to LPS (0.5 µg/mL), CBD (5 µM), THC (5 µM), or the aforementioned extracts (7 μg/mL). We found that the viability of THP-1 macrophages was not changed in response to any treatment . The celAs explained in the methods, after differentiation, macrophages were pre-treated with cannabinoids or cannabis extracts, and LPS was added 30 min later. Cell supernatants were evaluated for levels of cytokines 3 h after LPS exposure.LPS treatment drastically increased the levels of all cytokines. CBD pre-treatment significantly decreased the levels of IL-1β, while high CBD extract #131 did not A. Pre-trWe summarized our findings in We found that high THC extracts (#14 and #178) were more efficient than high CBD extract #131 in reducing the levels of IL-1β, IL-6, and TNF-α after LPS stimulation . In contThe Δ9-THC (Cat#T4764) and CBD (Cat#C-045) were purchased from Sigma-Aldrich . The 1 mg/mL stock solutions of cannabinoids were obtained by dissolving them in methanol and then keeping them at −20 °C.Lipopolysaccharide (Cat#L4391) was purchased from Sigma. The sterile PBS was used for dissolving LPS to obtain 1 mg/mL stock solution. Phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma-Aldrich . DMSO  was used to obtain 1 mg/mL stock solution. Then, the sterile PBS was used to obtain 5 μg/mL stock solution. Trypan Blue Solution (Cat#15250061), 0.4%, was purchased from Thermo Fisher Scientific .All cannabis plants were grown at the University of Lethbridge in the licensed facility. Cannabis sativa cultivars #14, #131, and #178 were used for the experiments. Plants were grown at 22 °C, 18 h light, 6 h dark, for a period of 4 weeks, with 4 plants per cultivar. To induce flowering, plants were shifted to chambers with 12 h light/12 h dark regime. Then the flowers were harvested and dried. Flowers from four plants were combined and then used for the extraction. Three grams of powdered plant tissue were used for the extraction of each studied cultivar. The prepared material was placed into a 250 mL Erlenmeyer flask, and then 100 mL of ethyl acetate was added to each flask. Next, flasks covered in tin foil were incubated overnight at 21 °C on a shaker at 120 rpm. The extracts were filtered and concentrated with the help of a rotary vacuum evaporator and then transferred to a tared 3-dram vial. The solvent was eliminated by evaporating to dryness in an oven overnight at 50 °C. The DMSO was used for the dissolution of crude extracts to obtain 60 mg/mL concentration, and extracts were kept at −20 °C. The complete culture medium was used to obtain the desired concentration of extracts (7 μg/mL). Before use, the extracts were filtered with 0.22 μm filter [v/v) ratio was used as the mobile phase of the HPLC. Calibration of standards and sample analysis were conducted using 2 μL of the injection volume. The detection of compound peaks was performed at 230 nm and 280 nm. For each cannabis cultivar, two samples with two technical replicates per treatment were analyzed [Agilent Technologies 1200 Series HPLC system was used for analyzing the levels of THC and CBD in each extract. Software ChemStation LC 3D Rev B.04.02  was used for the data acquisition, control of the instrument, and integration. A 9:1 methanol/chloroform  using 8610C GC coupled with a flame ionization detector . After calibration, the set of terpenes was used as standards (Restek Cat.#34095). LoQs and LoDs were set at less than 0.01%. Diagrams with retention times are available upon request. Two samples per cannabis cultivar were analyzed.2 at 37 °C. PMA  was used for the terminal differentiation of monocytes into macrophages. Next, cells were given fresh media for one day. The verification of successful transformation was conducted by evaluating cell adhesion and spreading under the light microscope [The THP-1 cells were purchased from American Type Culture Collection  . This sucroscope . 0.5 μg/croscope . CannabiTrypan blue assay was used for the determination of THP-1 macrophages’ viability and morphology. First, cells were washed with sterile PBS, then trypsinized. The fresh media was added to trypsinized THP-1 macrophages. After that, the cells were centrifuged at 1500 rpm for 5 min at 20 °C. The supernatant was discarded, and the pellet was resuspended in fresh media. The ratio of aliquot cells to trypan blue solution was 1:1. The automated cell counter, LUNA I (Logos Biosystems), was used for counting. The number of viable cells was determined by trypan blue exclusion. The results were shown as a percent of viability.g at 4 °C for 3 min prior to aliquoting to remove debris. The samples were stored at −70 °C. All prepared samples were shipped to Eve Technologies  on dry ice. Human Cytokine Array Proinflammatory Focused 13-plex (HDF13) discovery assay was used for measuring the levels of cytokines upon the first thaw. The results were analyzed using BioPlex 200 [Multiplex ELISA was performed for cytokine assessment. Cell culture supernatants were centrifuged at 3000× Plex 200 .p-value ˂ 0.05 was considered statistically significant.One-way analysis of variance (ANOVA), followed by Tukey post-hoc multiple comparison test, was performed in GraphPad Prism version 6.0 for windows, GraphPad software  . A p-valIn this work, we have established an LPS-induced model of the induction of inflammation in THP-1 macrophages. We found that the highest level of induction of pro-inflammatory cytokines was observed at 3 h post-exposure to LPS. We then demonstrated that pre-treatment with CBD, THC, or extracts high in one of these two cannabinoids substantially decreased the induction of cytokines after LPS treatment. CBD was more efficient than THC, and extracts were more efficient than single cannabinoids. IL-6, IL-8, and MCP-1 were most sensitive to pre-treatments with cannabinoids, while IL-1β, IL-10, and TNF-α were much less responsive.Several other models of inflammation were used in the past, including U937  and MonoMany studies have demonstrated the effects of single cannabinoids, such as THC and CBD, on inflammation . Other cIL-1β is the most prominent pro-inflammatory cytokine and plays a crucial role in inflammasome signaling . IL-6, aThe data we obtained on IL-1β levels was quite interesting. This was the only cytokine that decreased in response to CBD more efficiently than in response to high CBD extracts or high THC extracts. It remains to be shown whether this is a specific response to our extracts or the nature of the response of this cytokine to cannabinoids. It was also curious to note that CBD and THC had no effect on MCP-1 levels, while our extracts significantly downregulated high MCP-1 levels post-LPS. Our data and reports by others demonstrate that MCP-1 can indeed be downregulated by cannabis extracts . MCP-1 wAnother interesting result was the effect of cannabinoids on IL-10 levels. IL-10 is an anti-inflammatory cytokine that protects cells against profound inflammation. We observed that LPS induced this cytokine, and pure CBD as well as all extracts reduced it; in contrast, THC slightly increased it. Some studies have demonstrated that cannabinoids and cannabis extracts enhance the levels of anti-inflammatory cytokines. For example, in the murine model of bone marrow transplantation, it was demonstrated that pure THC and CBD, as well as cannabis extracts high in CBD and THC, reduced inflammation by reducing IL-17 secretion and enhancing IL-10 secretion . AnotherSince LPS was used to induce the inflammatory response, it was expected that it would also induce anti-inflammatory cytokines to counteract high levels of pro-inflammatory cytokines, which are required for cell survival. For effective clearance of pathogens, the pro-inflammatory response is crucial, while an excessive inflammatory response causes tissue damage. Hence, the human body always maintains equilibrium by activating counteractive pathways to bring down the pro-inflammatory response. Since levels of pro-inflammatory cytokines were elevated by LPS in our study, we observed a corresponding increase in IL-10 levels in the LPS group to counteract the increase in pro-inflammatory cytokines. We hypothesize that since our extracts and CBD were able to significantly downregulate the levels of several pro-inflammatory cytokines, they reduced the levels of IL-10, possibly to maintain equilibrium. On the other hand, THC was not able to reduce the levels of pro-inflammatory cytokines as effectively as CBD and, hence, did not change the high levels of IL-10 post-LPS. It is also possible that cannabis extracts inhibit all cytokines, regardless of their nature.We established the THP-1 macrophage model for the analysis of the anti-inflammatory properties of cannabinoids and cannabis extracts. We found that the studied cannabis extracts significantly prevented the increase in the levels of pro-inflammatory cytokines induced by LPS. It would be important in the future to treat cells with extracts after the induction of inflammation since, in general, there is a need to treat inflammation rather than prevent it. We may also want to compare the effects of extracts with those of well-known anti-inflammatory agents such as dexamethasone. For a better understanding of our results, it would be very beneficial to look at key transcription factors and target genes by performing transcriptome profiling, followed by analyses of relevant protein expression. The most important consideration in the future is to perform this study in vivo and confirm the immunomodulatory activity of the extracts.


The chemical derivatization to enhance the signal intensity and signal-to-noise (S/N) of several organophosphorus (OP) acids in liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) is illustrated. The OP class of compounds represents the environmental degradants of OP nerve agents and pesticides. N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) was utilized to derivatize a panel of eight acids consisting of five alkyl methylphosphonic acids  along with three dialkylphosphate analogs . The derivatization reaction with CAX-B was conducted in acetonitrile in the presence of potassium carbonate at 70 °C for 1 h. The resulting acid derivatives were analyzed with an LC-Orbitrap-ESI-MS/MS, and their dissociation processes were investigated. It was found that the derivatization procedure increased the limits of identification (LOIs) by one to over two orders of magnitude from the range of 1 to 10 ng/mL for the intact OP-acids to the range of 0.02–0.2 ng/mL for the derivatized acids utilizing an LC-MS(QqQ) in MRM mode, regardless of the sample matrix . The interpretation of the corresponding ESI-MS/MS spectra for each type of derivatized sub-OP family revealed the formation of characteristic neutral losses and a characteristic ion for the organophosphorus core. This derivatization is beneficial and useful for screening and identifying target and “unknown” OP acids. Organophosphorus (OP) nerve agents, such as V- and G-nerve agents, are extremely toxic compounds, and the threat they pose is well understood and recognized. These agents have relatively short lifespans in the human body and in the environment because they are hydrolyzed or metabolized to stable alkyl methylphosphonic acids (AMPAs) . BecauseDesignated laboratories involved in off-site analysis aim to enhance their analytical capabilities to meet the challenges associated with the verification of OP nerve agent indicators . This challenge becomes more difficult in the presence of high matrix interferences with low concentration levels of analytes . In addiTo overcome these limitations, various methods have been developed to enhance the sensitivity of these OP acids. Some of them include the use of dicationic or tricationic reagents ,17,18 poThis study is aimed to develop a qualitative technique to enhance the identification sensitivity of targeted environmentally relevant OP acids related to chemical warfare agents and pesticides utilizing chemical derivatization with N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) followed by LC-ESI-MS/MS. In addition, the interpretation of the mass spectral data and the determination of characteristic ions may assist both in sensitivity enhancement and the unambiguous identification of “unknown” OP acids.This study focused on the development of a generic and sensitive identification method for OP acids related to nerve agents and pesticides (ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), isobutyl methylphosphonic acid (IBMPA), cyclohexyl methylphosphonic acid (CMPA), pinacolyl methylphosphonic acid (PMPA), diethyl phosphate (DEP), dibutyl phosphate (DBP), and diethyl thiophosphate (DETP)) using an LC-ESI-MS/MS. In order to enhance sensitivity, the derivatization product of the OP-acids should possess a high proton-affinity moiety (cationic tag), which is highly responsive in positive ESI-MS mode. In addition, the derivatization product should fragment efficiently upon collision-induced dissociation (CID) and produce intense and diagnostic product ions for sensitive MS/MS detection and unambiguous identification. These diagnostic ions may be used for structural core-based screening methods. We chose to use the CAX-B derivatizing agent to generate precursor ions (possessing a cationic tag) and characteristic product ions (possessing a tropylium/benzyl cation), which are highly responsive in positive ESI-MS/MS mode. CAX-B was first studied by Wang et al.  and founThe conventional derivatization reaction of an OP-acid with a benzyl halide  for GC analysis  or LC-MSOrbitrap-ESI-MS/MS experiments were executed for the OP-acids after derivatization by varying the collision energy . Their ESI-MS/MS spectra were interpreted and enabled us to study the fragmentation pathways with the aim of seeking characteristic product ions indicative of the suborganophosphorus groups. The suggested product ion structures, depicted in m/z 328.2036) exhibited four product ions at m/z 227.0834, 199.0521, 121.0650, and 105.0700 . This ion is attributed to the loss of both the triethylamine and isopropylene, isobutylene, cyclohexylene, and 2,2-dimethyl-1-butylene in IMPA, IBMPA, CMPA and PMPA, respectively, and is indicative of the methylphosphonate core structure and provides information on the substructure of “unknown” OP acids.The Orbitrap-ESI-MS/MS spectra of the alkyl methylphosphonic acids after derivatization with CAX-B are illustrated in 105.0700 a. One ofm/z 227 in the ESI-MS/MS spectra of CAX-EMPA) was not observed in the ESI-MS/MS spectra of the CAX-B derivatives of IMPA, IBMPA, CMPA, and PMPA, due to the facile formation of two parallel dissociation processes resulting in trimethylamine and alkene loss. This is probably because alkyl substituents larger than ethyl tend to dissociate faster than ethyl substituents. This phenomenon was in accordance with our previous study on V-type nerve agents, where an O-Et cleavage was not observed in VX, but an O-iBu cleavage was observed in Russian VX m/z 358.2141, CAX-DBP, m/z 414.2768) revealed an ion attributed to the loss of a triethylamine from the [M + H]+ precursor ions to generate ions at m/z 257.0937 and 313.1566, respectively. In addition, ions were derived from the neutral losses of one and two ethylene groups in CAX-DEP (m/z 229.0624 and m/z 201.0310) and one and two butylene groups in CAX-DBP (m/z 257.0939 and 201.0311). The ion at m/z 201.0311 is indicative of the dialkyl phosphate core structure and provides information on the substructure of “unknown” OP acids.These three acids contain two ethyl chains  or two butyl chains (DBP) attached to oxygen. As assumed, the ESI(+)-MS/MS spectra of the derivatives  and m/z 217.0084 (loss of two ethylene groups) were also observed. The ion at m/z 217.0084 is characteristic of the dialkyl thiophosphate core structure and is vital for determining the structure of “unknown” OP acids of the dialkyl thiophosphate substructure. Additionally, a low abundance, diagnostic ion at m/z 109.0050 [C2H6O3P]+ was observed. This ion is attributed to the monoethyl phosphate cation. The dissociation of two consecutive alkyl chains was in accordance with the experimental findings concerning fragmentations of phosphates and reported in the literature [The ESI(+)-MS/MS spectrum of DETP after derivatization with CAX-B revealed a high abundance ion attributed to the loss of triethylamine from the [M + H]terature ,28.To compare the identification sensitivity between the intact OP acids and the CAX-B-derivatized OP acids, OP acids at concentrations of 10 ng/mL were derivatized with CAX-B to the corresponding OP derivatives, and the reaction mixtures were diluted 1:5 with water and analyzed with an LC-ESI-MS/MS in MRM mode. For the nonderivatized OP acids, the most intense MRM transition was observed with a signal-to-noise ratio (S/N) of 20–60. However, for CAX-OP-acid derivatives, two to five high-intensity MRM transitions were observed, with a signal-to-noise ratio (S/N) of 1000–3000. The MRM transition signal with the highest S/N ratio of the intact EMPA ((−)123 > 95) was 60 times lower than that of the MRM transition signal with the highest S/N ratio of the CAX-EMPA ((+)328 > 199) using an LC-ESI-MS/MS a,b, and In conclusion, CAX-B was found to be a powerful reagent for the derivatization of OP acids. Several OP-acid derivatives presented the production of an analyte—characteristic of the first product ion (as a xylyl-based cation) from the loss of neutral triethylamine from the precursor ion .+. After derivatization of the OP acids with CAX-B, these O-alkyl cleavages were also observed; however, at a higher intensity due to the attached xylene-tag moiety, both in the precursor and product ions. In addition, the formation of the OP core characteristic ion is beneficial for the determination of the specific substructure (phosphonate/phosphate/thiophosphate) and is useful for the identification of “unknown” OP acids.O-alkyl cleavages were observed in all intact OP acids in positive ion mode, generating several sequential product ions from the precursor ion [M + H]Method evaluation was performed in ACN. As this method is qualitative only, method performance was determined by evaluating the calibration curve linearity, limits of identification (LOIs), relative standard deviations (RSDs), and stability of CAX-OP-acid derivatives.2) greater than 0.99, as shown in The derivatization reaction for all OP acids was carried out in ACN in the concentration range of 0.01–10 ng/mL. All the calibration curves for the CAX-OP-acid derivatives were linear over the range of 0.01–10 ng/mL , with a coefficient of determination . The repeatability of the method was determined by conducting three independent derivatizations at each concentration in the calibration curve and measuring the resulting relative standard deviations (RSDs) in the intensity of the most intense MRM signal for each analyte. For all seven CAX-OP acids, the RSDs were lower than 20%.After demonstrating the ability of the method to convert OP acids into their corresponding CAX-OP esters, we tested its performance on real-world matrices. The reactivity of CAX-B was assessed toward OP acids in acetonitrile extracts of plant, concrete, and hair, which represent environmental and clinical matrices that OP nerve agents and pesticides may degrade in, resulting in low amounts of OP-acids.2) greater than 0.99  and to affect the derivatization rates in LC–MS analysis. Matrix effects were evaluated by comparing the acetonitrile extracts of a blank matrix spiked with OP-acids and pure acetonitrile solvent, followed by derivatization with CAX-B prior to analysis. Acetonitrile extracts of plants, hair and concrete were spiked with five OP acids  at concentrations between 0.02 and 10 ng/mL, and the derivatization procedure was applied. The linearity and matrix effects were evaluated. The calibration curves for the derivatization product were linear in the range of 0.02–10 ng/mL, with a coefficient of determination . Formic acid (MS grade), acetonitrile (MS grade), and water (MS grade) were obtained from Biolab .Pooled human head hair was collected from a barber shop, plant leaves were collected from lemon and orange trees, and a concrete block was collected from a construction site.A stock solution (100 ng/mL) of EMPA, IMPA, IBMPA, CMPA, PMPA, DEP, DBP, and DETP was prepared in acetonitrile (ACN) and stored at −20 °C. This solution was further diluted in acetonitrile/acetonitrile extracts prior to derivatization to concentrations of 0.01–10 ng/mL.2CO3 and 0.5 mg of the derivatizing agent CAX-B were added into an Eppendorf tube containing 500 μL of an acetonitrile/acetonitrile extract containing 5 pg to 5 ng of analytes. Then, the solution was stirred using an Eppendorf vortex mixer at 70 °C and 800 rpm for 1 h. The content was then diluted 1:5 with water and transferred into an LC conic vial prior to LC-ESI-MS/MS analysis.First, 0.5 mg of KPlant leaves (5.2 g), human hair (2.2 g), and concrete (20.2 g) were vortexed with 28, 17, and 20 mL of acetonitrile, respectively, in a 50 mL polypropylene tube for 30 s and rested in a fume hood at room temperature for 85 min. Then, the acetonitrile extracts were filtered using a 0.22 µm PVDF filter into a fresh glass bottle and kept at 4 °C.Each acetonitrile extract (500 µL) was spiked with the analytes and immediately subjected to the abovementioned derivatization procedure.Chromatographic separation was achieved using a reversed-phase column  at 40 °C with a flow rate of 0.3 mL/min. All injections were 5 μL.For the separation of intact OP acids, the mobile phase consisted of 1 mM ammonium formate in water (A) and in methanol (B). For the separation of CAX-OP derivatization products, the mobile phase consisted of 0.1% formic acid in water (A) and methanol (B).The gradient profile began with 100% mobile phase A and ramped linearly to 5% A over 6 min. Then, the flow was held at 5% A for 3.5 min, switched back to 100% A, and held for 5.5 additional minutes up to a total cycle time of 15 min to equilibrate the column for the next sample.The LC-MS/MS system consisted of an Acquity UPLC I class system coupled to a Xevo TQ-S triple quadrupole tandem mass spectrometer , which was operated with a positive/negative ESI source in multiple reaction monitoring (MRM) mode. The MS/MS parameters were set as follows: capillary voltage, 0.6 kV; source temperature, 150 °C; desolvation temperature, 500 °C; desolvation gas flow, 800 L/h; cone gas flow, 150 L/h; and collision gas flow, 0.15 mL/min. The Intellistart feature in Waters MassLynx software  was used for determining the most intense MRM transitions and optimizing the cone voltages and collision energies.+ with a relatively abundant product ion at m/z 97, the OP acids yielded deprotonated molecules [M − H]− with the two most abundant MRM transitions listed in The OP acids related to the nerve agents EMPA, IBMPA, CMPA, and PMPA were analyzed in their intact forms to determine their responses with an RPLC-ESI(−/+)-QqQ-MS/MS before derivatization. Except for EMPA that yielded a protonated molecule [M + H]+ with the two most abundant MRM transitions listed in The OP acids related to the pesticides DEP, DBP, and DETP were also analyzed in their intact forms to determine their responses with an RPLC-ESI(+)-QqQ-MS/MS before derivatization. These OP acids yielded protonated molecules [M + H]The detection and identification of all OP-acids after derivatization with CAX-B was performed in positive ion MRM mode using the most abundant MRM transitions listed in The LC-MS system consisted of an Agilent 1290 HPLC system  connected to a Q-Exactive+ Orbitrap mass spectrometer  equipped with a heated electrospray ionization (HESI) ion source operated in positive ion mode.The ESI ion source operating parameters were as follows: electrospray voltage was 1.25 kV; sheath gas flow rate was 45 (arbitrary units); auxiliary gas was 10 (arbitrary units); sweep gas was 2 (arbitrary units); auxiliary gas heater temperature was 400 °C; and the capillary temperature was 275 °C. MS and MS/MS parameters are listed in OP acid solutions in ACN at a concentration range of 0.01–10 ng/mL were analyzed prior to derivatization in the negative and positive ion modes and after derivatization in the positive ion mode. The S/N ratios of the two most intense MRM transitions before and after derivatization were compared.CAX-B was found to be an efficient derivatizing agent for increasing the sensitivity of all investigated OP acids in tandem mass spectrometry from complex matrices, such as concrete, plant, and hair extracts. The conversion of the OP acids to their corresponding derivatives improved the detection sensitivity by one to over two orders of magnitude compared with the detection of non-derivatized analytes in LC-ESI-MS/MS analysis and allowed for detection in the low ppt range. Moreover, its ease of use and the formation of product ions characteristic of the sub-OP group make it a good choice for the enhanced detection of targeted and “unknown” OP acids.


Low back pain is one of the most common complaints affecting many individuals. The McGill Pain Questionnaire is used in various clinical settings to assess different types of pain and one of the most extensively used outcomes measures for pain in the world. The purpose of this study was to translate and validate the original English version of the SF-MPQ-2 into Urdu (SF-MPQ-2-U).For this study, Mapi Research Trust protocols were followed for the forward and backward translation. Test-retest reliability was used to assess the reliability. Cronbach’s alpha and Omega was used to determine internal consistency. Pearson’s correlation was used to evaluate convergent validity. Confirmatory factor analysis was also conducted.The Cronbach’s alpha for SF-MPQ-2-U was 0.73 to 0.79, indicating acceptable internal consistency. Omega score for the SF-MPQ-U were 0.918. The ICC varied from 0.799 to 0.878 for domains of SF-MPQ-2-U. The CFA of the SF-MPQ-2-U met model fit indices with GFI and NFI > 0.90. The inter-scale correlation between baseline and re-test data was from 0.63 to 0.71, indicating a positive and strong correlation. The SF-MPQ-2-U and ODI-U had a baseline correlation of 0.547. The correlation of SF-MPQ-2-U & VAS at baseline data was 0.558. Pearson’s correlation between subscales was r = 0.253 with p 0.01, which was statistically significant.The SF-MPQ-2-U is considered to have good convergent validity at inter scale and between two scale levels. Reliability was checked by test-retest reliability, Internal consistency was checked using Cronbach’s alpha and Omega that showed good internal consistency for measuring different types of pain in patients with low back pain who speak Urdu. To make the questionnaire more valid and reliable, it is recommended for the researchers to do in-depth research on larger sample size. Pain is one of the most prevalent sensations of discomfort among every age group, regardless of male or female, and can cause sleep issues and psychological distress in the patient. Chronic pain is one of the reasons that causes people to get medical care linked to functional limitations . Pain caLow back pain is one of the most common complaints among the elderly population and is experienced by 80% of the population at least once in their lifetime. It has adverse effects on the quality of life. If the pain persists for more than 12 weeks, it is known as chronic pain. It can be due to impaired physical functioning, clinical anxiety, depression, lumbar stenosis, spine fracture, inflammatory disease, or nerve root compression –8.The elderly population has been cared for through the proper healthcare system of the country. These issues place a strain on the healthcare system as well as the economy of the country. The elderly’s utilization of healthcare services is influenced by various factors, including socioeconomic, cultural, financial, and geographical resource availability .Several scales are used to assess pain intensity which include: Numerical Pain Rating Scale, Visual Analogue Scale, Verbal rating scales, Face Rating Scale,  McGill PEach of these tools is a credible measure of pain and has undergone test-retest reliability. The Oswestry Disability Index (ODI) is one of them and is a highly reliable tool for assessing disability and pain in people suffering from low back and lumbar radiculopathy. It is one of the most highly regarded measures for determining the quality of life and level of disability in patients with lumbar radiculopathy .Both The NPRS and the VAS scale are one-dimensional instruments that solely assess pain severity. However, The McGill Pain Questionnaire (MPQ) is a multidimensional questionnaire that includes information regarding affective, sensory and evaluative aspects of pain perception. The McGill Pain Questionnaire MPQ was created in 1975 and has quickly become one of the most extensively used questionnaires globally. However, it was the questionnaire is long and is one of the lengthy and time-consuming for patients to complete questions . In 1987Urdu is the most widely spoken language in Pakistan. It is the language that medical professionals use when educating patients and their families about their medical issues. As a result, it is critical to translate the English version of the reliable and valid SF-MPQ-2 questionnaire into Urdu for use among people who speak Urdu as their first language living all across the world. Therefore, the purpose of this study was to translate and validate the English version of the SF-MPQ-2 into Urdu (SF-MPQ-2-U) so that it can be used in research and clinical settings to assess pain.The 22 components of SF-MPQ-2 are used to measure several types of pain, including affective, neuropathic, intermittent, and continuous pain . There aThe ODI is a self-reported outcome measure that assesses low back pain . There aTwo local translators translated the English version into the Urdu language. Each of the translators produced an independent Urdu translation (T1 and T2). Another version, V3, produced by the translators and the principal investigator, removed any ambiguities and chose the most appropriate translations from T1 and T2.Two translators who were unaware of the English version carried out the backward translation of the V3. They translated the Urdu version (V3) into the Source language (BT1). The purpose of this phase was to make sure that the target language gives the same content that is used in the source language.After completing the backward translation (BT1), the local translator compared the backward translated English version and the original English version SF-MPQ to rule out any misunderstandings. After analyzing BT1 and Source language, they removed any ambiguities and chose the most appropriate translation.To check the understandability of the pre-final version, a pilot study was done on 15 male and female patients (aged 30–60 years) with back pain who were native speakers of the target language.All the participants with structural abnormalities, any disability, language and memory problems, and who could not read and understand the Urdu language were excluded from the study. The pre-final version was distributed among them, and they were interviewed, patients were asked if they felt there was any difficulty reading or understanding any item. After the mutual discussion of both translator and backward translator, this version was then finalized and chosen to be distributed among the participants as the final translated version.An expert committee consisting of medical professionals, Ph.D. Scholars and translators checked for typing mistakes, grammatical errors, and spelling mistakes. No typing, spelling mistakes, or grammatical errors were found in V3. Therefore, the expert committee finalized the translated version 3 (V-3) and approved its distribution to test psychometric properties.The non-probability convenient sampling strategy was used to select 110 patients. The sample size was calculated according to the guidelines provided by Nicolaou et al., the sample size should be bigger than the number of variables; N > p, which is true in our instance. Gorsuch et al. suggested using 5 cases per indicator. Although any of these ratios—5:1, 10:1, and 20:1—are suitable, 10:1 is the most frequently used. Keeping in mind the methodology of our study, 5:1 was the most suitable option for our study [The SF-MPQ-2-U and ODI-U and VAS were provided to the patients. A standardized self-administration procedure was followed. Instructions were given verbally to each patient. The translation process started after receiving the ethical approval from the institutional review board. The data collection and patient consent were under the guidelines of Declaration of Helsinki. The translation procedure was governed by the protocol defined by MAPI e-trust services, the original owners of the SF-MPQ-2.SPSS version 25 was used to evaluate the data collected from the participants. SF-MPQ-2-U ODI-U and VAS descriptive statistics were analyzed using means, standard deviations, and frequencies. The normality of data was assessed by using the Shapiro-Wilk test.Convergent Validity, Pearson’s Correlations, ICC and Test-Retest Reliability:The relationship between SF-MPQ-2-U, ODI-U, and VAS was evaluated for convergent validity. The convergent validity of SF-MPQ-2-U was examined by investigating the correlation between inter-scales. The strength of the correlation between the two domains was determined using Pearson’s correlation coefficient (r) The SF-dependability was evaluated using internal consistency and test re-test reliability. The internal consistency of SF-MPQ-2-U for the total scale was assessed using Cronbach’s alpha and McDonald’s Omega. The intraclass correlation coefficient (ICC), which ranges from 0 to 1, was used to determine test-retest reliability. In order to determine the test-retest reliability, data were collected twice with a gap of 48 h. No intervention was applied to the participants during these 48 h. Internal consistency was checked using Cronbach’s alpha Low ICC numbers (0.5) indicate poor reliability, moderate ICC values (0.5–0.75) show moderate reliability, good ICC values (0.75–0.9) indicate good reliability, and any value above 0.9 indicates great reliability .Confirmatory factor analysis of the data was performed using AMOS software, with the goodness of fit index, normed compliance index, and mean square root of estimated errors as the evaluation measures. Maximum likelihood estimation method was used in this analysis. Model fit of the confirmatory factor analysis (CFA) was assessed using the following fit indices: Goodness of Fit index , Normed Compliance Index , Root Means Square Error of Approximation , ComparOut of 110 participants, forty-three were males (39.1%), and 67 were females (60.9%). The mean age of participants was 45.16 years, with a standard deviation of ± 7.58. Out of the total sample size selected for the study, 33(30%) participants were between the age of 31–40 years, 48 (43.6%) participants were between 41 and 50, and 29 (26.4%) participants were between 51 and 60 years. The participants of the study were also categorized according to their socioeconomic background. Out of 110 total participants, two (1.8%) were from the lower class, 85 (77.3%) were from the middle class, and 23(20.9%) were from the upper class.  internal consistency of SF-MPQ-2-U of baseline and retest data throughout its four domains  for the Continuous, Intermittent, Neuropathic, and Affective domains were 0.811 (CI = 0.755–0.859), 0.878 CI = 0.842–0.909), 0.799 (CI = 0.738–0.850), and 0.875 (CI = 0.837–0.908), respectively, which is highly correlated and indicating good reliability  with persistent low back pain, aged 30 to 60, who could speak and understand Urdu. The average age was 45.16 years, similar to previous studies. The SF-MPQ-2 was translated using a sample size of 194, and previous studies also had similar sample sizes and demographics. This information highlights the comparability of our study to previous research.  internal consistency. Cronbach’s alpha for previous studies ranged from 0.8 to 0.9 , 18. DurIntraclass correlation coefficients with a 95% confidence interval demonstrated acceptable reliability. In case of these results of ICC demonstrated that the SF-MPQ-2-U has an acceptable internal consistency and test-retest reliability. ICCs in prior investigations were 0.973 and 0.027, showing strong reliability. The previous literature showed that the translated versions of SF-MPQ-2 after translation remain valid and reliable compared to the original questionnaire , 18.One method used to test SF-MPQ-2-U validity was by assessing its convergent validity. In this study, the ODI-U and VAS scales and the SF-MPQ-2-U were utilized to assess convergent validity. VAS, ODI, SF-36, and WOMAC questionnaires have already been utilized in the literature for convergent validity for the translated SF-MPQ-2 versions , 26, 27 Pearson’s correlation between scales was r = 0.253, with a significance level of p < 0.01. Previously, the SF-MPQ-2 was significantly correlated with other scales used to evaluate convergent validity . The PeaThe strong positive correlation (p = 0.01) between the SF-MPQ-2-U domains and the ODI-U and VAS components was already presented in the results. However, it is important to note that the SF-MPQ-2-U and ODI-U subscales, except for the intermittent domain of the SF-MPQ-2-U with Walking and Traveling, were positively correlated. The Affective domain of the SF-MPQ-2-U showed moderate correlation values ranging from 0.25 to 0.28 with standing, sleeping, and social life, and it was negatively related to social life (p < 0.01). These findings are in line with previous research that has shown similar relationships between pain measures and disability scales , Continuous and Neuropathic variables r = 0.635), Continuous and Affective (r = 0.639), Intermittent and Neuropathic variables (r = 0.717), intermittent and affective variables  and between Neuropathic and Affective variables (r = 0.655). The effect size of these correlations was good at  respectively , the goodness of fit indices indicate that the model fits the data moderately well. The goodness of fit index (GFI) was 0.805, indicating that 80.5% of the variance in the observed variables was explained by the model. The adjusted goodness of fit index (AGFI) was 0.757, indicating that the model fit the data reasonably well. The normed fit index (NFI) was 0.686, indicating a moderate fit between the model and the data. The comparative fit index (CFI) was 0.858, indicating a good fit between the model and the data. The Tucker-Lewis index (TLI) was 0.838, indicating a moderate fit between the model and the data. The root mean square error of approximation (RMSEA) was 0.070, indicating a reasonable fit between the model and the data. Finally, the expected cross-validation index (ECVI) was 3.769, which is a measure of the model’s predictive accuracy. Overall, the results suggest that the CFA model has a moderately good fit to the data, but there may be some room for improvement. Figure


Preterm birth (PTB) is a complex and significant challenge in obstetrics. Thus, clinicians and researchers have paid a keen interest in the identification of women at a high risk for PTB. This study aimed to develop a PTB risk assessment scale based on the preliminary 32-item Preterm Birth Risk Assessment Scale-Korean version (PBRAS-K).+0 weeks after experiencing preterm symptoms and were admitted to high-risk pregnancy maternal–fetal intensive care units (MFICUs). After an item-reduction process in the exploratory factor analysis, the psychometric property scales were assessed using SPSS Statistics version 27.0, and the confirmatory factor analysis was conducted using AMOS version 27.0.We enrolled 298 participants  who delivered before 372 test of sphericity confirmed the adequacy of the sample for factor analysis (KMO = .81 (> .80), χ2 = 1841.38, p < .001). The final version of the PBRAS-K comprised 23 items within seven dimensions. Factor analysis identified items explaining 65.9% of the total variance. The PBRAS-K achieved a mean score of 35.58 (± 10.35) and showed high internal consistency and satisfactory reliability . Regarding concurrent validity, the PBRAS-K exhibited a low-to-moderate correlation with the PTB risk . As for criterion validity and convergent validity, the PBRAS-K showed a positive and high correlation with the Somatic Awareness Scale with Spontaneous Preterm Labor (SPL-SAS)  and pregnancy-related stress , respectively. Risk scoring for preterm delivery and SPL-SAS were moderately correlated .The Kaiser–Meyer–Olkin (KMO) test and Bartlett’s χPBRAS-23-K is a valid and reliable instrument for assessing the risk for PTB in pregnant women. Clinical nurses are encouraged to apply and obtain information regarding effective interventions in MFICUs. This scale provides meaningful results and reflects the opinions of women who had experienced PTB. The PBRAS-23-K should be evaluated for standardization and cut-off scores using larger sample sizes in the future.The online version contains supplementary material available at 10.1186/s12884-023-05975-x. Preterm birth (PTB), defined as the birth of a baby at <37 weeks of gestation , 2, is tSeveral clinicians have attempted to predict the incidence of PTB; nonetheless, PTB remains difficult to predict. According to the American Association for Clinical Chemistry guidelines, biomarkers such as interleukin-6, placental alpha macroglobulin-1, and fetal fibronectin have low positive predictive values and provide limited utility in the diagnostic algorithms most commonly applied in the United States . ApproxiPTB prediction involves a screening test with high sensitivity and high negative predictive value. Various screening tools can be classified into four groups—namely, monitoring of uterine activity, assessment of cervical length, measurement of cervical fetal fibronectin, and detection of the presence of bacterial vaginosis in early pregnancy . While tTherefore, a scale that can inform clinicians and pre-pregnant women about the causes of PTB must be developed to obtain proper care. Based on the nine components of PTL and PTB , the autThis methodological study adopted a cross-sectional survey design to evaluate the reliability and validity of the PBRAS-32-K  accordin+0 weeks, and (ii) pregnancy with no symptomatic problems. The offline survey was administered in an outpatient setting. It was limited to outpatient surveys because there was a concern that answering the questionnaire could be stressful for hospitalized women and that this could lead to increased preterm labor. The offline survey was conducted at hospitals with MFICUs across the country.Offline data collection was planned for antepartum women who had experienced PTL at each maternal-fetal intensive care unit (MFICU) center; however, this was changed to an online Google platform because of the coronavirus disease pandemic. One hundred forty participants completed a questionnaire at the outpatient clinic of the hospital, and others completed it on an online platform. With the cooperation of the heads of seven MFICUs and head nurses, a recruitment notice was posted on the bulletin board. Pregnant women responded to the questionnaire using QR codes. The participants provided informed consent for their participation on the platform, verifying that they understood the purpose and content of the study. The required sample size was over 300, or five to ten times the number of exploratory factor analysis (EFA) items and five times the number of final confirmatory factor analysis (CFA) items . The res® SPSS® Statistics 27TM, and IBM® SPSS® 27TM  was used for model fitting. Descriptive statistics were used to determine the frequency, range, mean, and standard deviation of the participants’ demographic and clinical characteristics. All other tests were two-tailed, and a p-value of <5% was considered to be statistically significant. Item analysis included mean and standard deviation, skewness and kurtosis, and corrected item-total correlation coefficient. The absolute skewness and kurtosis values were evaluated to determine whether they were less than 3.0 for skewness and less than 7.0 for kurtosis, satisfying the item analysis conditions. Data were automatically analyzed using SPSS version 27.0. Additionally, the total scores and intra-item correlations were analyzed to determine whether the values were ≥.30  , the internal consistency of the PBRAS-23-K had a corrected item-total correlation of over .30. Nonetheless, even if these five items were deleted, Cronbach’s alpha did not increase above .85, and reliability of the subscales was .60–.83, except for factor 6 (.44) and factor 7 (.20). Therefore, all 23 items were retained in this study , indicating the high validity of a similar construct. With respect to concurrent validity, the PBRAS-23-K exhibited a low-to-moderate positive correlation [r = .45, p < .001). As for criterion validity, SPL-SAS was evaluated for somatic symptoms, and PBRAS-23-K had a high correlation coefficient  ; These values satisfied cutoff criteria for fit indices [Other factors associated with PTB should also be considered. In 2020, Kim visualized nine components explaining PTB . In thisPregnant women with PTL are at risk for giving birth prematurely; consequently, they are hospitalized and managed in MFICUs. In MFICUs, patients are required to rest, and an anti-contraction drug is administered to prevent labor progression. The patients are discharged when uterine contractions disappear. While admitted, women with PTL are not concerned about the hard work that they perform at home (or workplace) or how stressed they were. When they go home, they are again in a similar stressful situation, and many return to the hospital within a few days.r < 0.3  for broad measurements. As suggested by Clark and Watson in 1995, the item-total correlation items (0.15–0.20) should be selected for broad measurements, and items with >0.40 are recommended for narrow measurements [r = .57, p < .001) and exhibited a low-to-moderate positive correlation [r = .45, p < .001). Rea and Parker reported that the r-value (0.4–0.6) indicated a moderately strong positive relationship [r = .65, p < .001). Except for Q28 (r = .48), a particular construct correlated with other tests that assessed the same construct (≥.50) but did not correlate with tests that measured different constructs (<.33). In other words, the PBRAS-23-K had high convergent and concurrent validity.In this study, the internal consistency showed a Cronbach’s alpha of .85 for the total items, indicating an adequate instrument . Furtherurements . In the urements , the PBRrelation  with RPDtionship ; howeverThe PBRAS-23-K exhibits a partial correspondence with the seven factors elucidated using the generalized methodology with machine learning modelling as reported by Della Rosa et al. . The ratIn this study, the researcher adopted a minimum sample size of five times the number of items for the CFA based on a previous study . In thisThe PBRAS-23-K was developed using seven factors based on a previous study, which reported that PTB had nine components. This scale should be further simplified for clinical and public nurses to counsel, educate, and provide care to women at risk for PTB. The author is currently developing a PTB risk assessment App using the items from PBRAS-K.The PBRAS-23-K is a valid and reliable instrument for assessing the risk for PTB in pregnant women. Clinical nurses and obstetricians should be encouraged to obtain information regarding effective interventions in MFICUs. This scale provides meaningful results and reflects the opinions of women who had experienced PTB. In the future, the scale should be evaluated for standardization and cut-off scores using larger sample sizes and is suggested to be used for the components of PTB modeling.Additional file 1:Table S1. Preterm Birth Risk Assessment Scale-Korean version (PBRAS-23-K). Additional file 2:Table S2. Internal consistency of each factor.Additional file 3:Figure S1. Confirmatory factor analysis.